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Association of a Methanol Extract of Rheum undulatum L. Mediated Cell Death in AGS Cells with an Intrinsic Apoptotic Pathway.

Hong NR, Park HS, Ahn TS, Jung MH, Kim BJ - J Pharmacopuncture (2015)

Bottom Line: Also, treatment with MERL increased the activities of caspase-3 and -9 compared with the control.However, inhibitions of p38, extracellular signal regulated kinases (ERKs) and C-Jun N-terminal kinases (JNK) by MERL treatment did not affect cell death.These results suggest that MERL mediated cell death is associated with an intrinsic apoptotic pathway in AGS cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Longevity and Biofunctional Medicine, Healthy Aging Korean Medical Research Center, School of Korean Medicine, Pusan National University, Busan, Korea.

ABSTRACT

Objectives: Rheum undulatum L. has traditionally been used for the treatment of many diseases in Asia. However, its anti-proliferative activity in cancer has still not been studied. In the present study, we investigated the anti-cancer effects of methanol extract of Rheum undulatum L. (MERL) on human adenocarcinoma gastric cell lines (AGS).

Methods: To investigate the anti-cancer effect of MERL on AGS cells, we treated the AGS cells with varying con¬centrations of MERL and performed 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. Cell cycle analyses, measurements of the mitochondrial membrane potential (MMP), caspase activity assays and Western blots were conducted to determine whether AGS cell death occurred by apoptosis.

Results: Treatment with MERL significantly inhibited growth of AGS cells in a concentration dependent manner. MERL treatment in AGS cells leaded to increased accumulation of apoptotic sub G1 phase cells in a concentration dependent manner. In control cultures, 5.38% of the cells were in the sub G1 phase. In MERL treated cells, however, this percentage was significantly increased (9.95% at 70 μg/mL, 15.94% at 140 μg/mL, 26.56% at 210 μg/mL and 38.08% at 280 μg/mL). MERL treatment induced the decreased expression of pro-caspase-8 and -9 in a concentration dependent manner, whereas the expression of the active form of caspase-3 was increased. A subsequent Western blot analysis revealed increased cleaved levels of poly (ADP-ribose) polymerase (PARP) protein. Also, treatment with MERL increased the activities of caspase-3 and -9 compared with the control. MERL treatment increased the levels of the pro-apoptotic truncated Bid (tBid) and Bcl2 Antagonist X (Bax) proteins and decreased the levels of the anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein, whose is the stabilization of mitochondria. However, inhibitions of p38, extracellular signal regulated kinases (ERKs) and C-Jun N-terminal kinases (JNK) by MERL treatment did not affect cell death.

Conclusion: These results suggest that MERL mediated cell death is associated with an intrinsic apoptotic pathway in AGS cells.

No MeSH data available.


Related in: MedlinePlus

Effects of MERL on the MMP values and the levels of tBid, Bcl-2 and Bax proteins in AGS cells. (A) Cells were treated with the indicated concentration of MERL for 24 hours. Cells were collected and incubated with JC-1 (10 μM) for 20 minutes at 37˚C in the dark. The cells were the washed once with PBS and analyzed by a DNA flow cytometer. (B) The cell lysates obtained from cells grown under the same conditions as (A) were separated by SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were probed with the indicated antibodies. Actin was used as an internal control. Bars represent the mean ± S.D. *P < 0.01. MERL, methanol extract of Rheum undulatum L.; MMP, mitochondrial membrane potential; tBid, truncated Bid; Bcl-2, B-cell lymphoma 2; Bax, Bcl2 Antagonist X; AGS, adenocarcinoma gastric cell lines; JC-1, 5,5′, 6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide; PBS, phosphate buffered saline; SDS, sodium dodecyl sulfate; S.D., standard deviation.
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Figure 004: Effects of MERL on the MMP values and the levels of tBid, Bcl-2 and Bax proteins in AGS cells. (A) Cells were treated with the indicated concentration of MERL for 24 hours. Cells were collected and incubated with JC-1 (10 μM) for 20 minutes at 37˚C in the dark. The cells were the washed once with PBS and analyzed by a DNA flow cytometer. (B) The cell lysates obtained from cells grown under the same conditions as (A) were separated by SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were probed with the indicated antibodies. Actin was used as an internal control. Bars represent the mean ± S.D. *P < 0.01. MERL, methanol extract of Rheum undulatum L.; MMP, mitochondrial membrane potential; tBid, truncated Bid; Bcl-2, B-cell lymphoma 2; Bax, Bcl2 Antagonist X; AGS, adenocarcinoma gastric cell lines; JC-1, 5,5′, 6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide; PBS, phosphate buffered saline; SDS, sodium dodecyl sulfate; S.D., standard deviation.

Mentions: Mitochondria, which are specialized organelles, play a critical role in apoptosis. Intermembrane spaces of mitochondria contain many pro-apoptotic proteins including cytochrome c. Disruption of the outer mitochondrial membrane, which can be caused by several events, results in the release of cytochrome c, leading to the activations of caspase-9 and effector caspases, which eventually causes apoptotic cell death [16-19]. For this study, the effects of MERL on the levels of MMP were measured by using a flow cytometer with a JC-1, mitochondrial specific probe. As shown in Fig. 4a, MERL treatment increased the loss of MMP in a concentration dependent manner, indicating that treatment with MERL causes depolarization of the mitochondrial membrane. We next determined the changes in the levels of the anti- or the pro-apoptotic proteins. As indicated in Fig. 4b, MERL treatment increased the levels of the pro-apoptotic tBid and Bcl2 Antagonist X (Bax) proteins and decreased the levels of the anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein. These results suggest that MERL treatment induces mitochondrial dysfunction via decreased levels of Bcl-2 and increased levels of tBid and Bax.


Association of a Methanol Extract of Rheum undulatum L. Mediated Cell Death in AGS Cells with an Intrinsic Apoptotic Pathway.

Hong NR, Park HS, Ahn TS, Jung MH, Kim BJ - J Pharmacopuncture (2015)

Effects of MERL on the MMP values and the levels of tBid, Bcl-2 and Bax proteins in AGS cells. (A) Cells were treated with the indicated concentration of MERL for 24 hours. Cells were collected and incubated with JC-1 (10 μM) for 20 minutes at 37˚C in the dark. The cells were the washed once with PBS and analyzed by a DNA flow cytometer. (B) The cell lysates obtained from cells grown under the same conditions as (A) were separated by SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were probed with the indicated antibodies. Actin was used as an internal control. Bars represent the mean ± S.D. *P < 0.01. MERL, methanol extract of Rheum undulatum L.; MMP, mitochondrial membrane potential; tBid, truncated Bid; Bcl-2, B-cell lymphoma 2; Bax, Bcl2 Antagonist X; AGS, adenocarcinoma gastric cell lines; JC-1, 5,5′, 6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide; PBS, phosphate buffered saline; SDS, sodium dodecyl sulfate; S.D., standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 004: Effects of MERL on the MMP values and the levels of tBid, Bcl-2 and Bax proteins in AGS cells. (A) Cells were treated with the indicated concentration of MERL for 24 hours. Cells were collected and incubated with JC-1 (10 μM) for 20 minutes at 37˚C in the dark. The cells were the washed once with PBS and analyzed by a DNA flow cytometer. (B) The cell lysates obtained from cells grown under the same conditions as (A) were separated by SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were probed with the indicated antibodies. Actin was used as an internal control. Bars represent the mean ± S.D. *P < 0.01. MERL, methanol extract of Rheum undulatum L.; MMP, mitochondrial membrane potential; tBid, truncated Bid; Bcl-2, B-cell lymphoma 2; Bax, Bcl2 Antagonist X; AGS, adenocarcinoma gastric cell lines; JC-1, 5,5′, 6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide; PBS, phosphate buffered saline; SDS, sodium dodecyl sulfate; S.D., standard deviation.
Mentions: Mitochondria, which are specialized organelles, play a critical role in apoptosis. Intermembrane spaces of mitochondria contain many pro-apoptotic proteins including cytochrome c. Disruption of the outer mitochondrial membrane, which can be caused by several events, results in the release of cytochrome c, leading to the activations of caspase-9 and effector caspases, which eventually causes apoptotic cell death [16-19]. For this study, the effects of MERL on the levels of MMP were measured by using a flow cytometer with a JC-1, mitochondrial specific probe. As shown in Fig. 4a, MERL treatment increased the loss of MMP in a concentration dependent manner, indicating that treatment with MERL causes depolarization of the mitochondrial membrane. We next determined the changes in the levels of the anti- or the pro-apoptotic proteins. As indicated in Fig. 4b, MERL treatment increased the levels of the pro-apoptotic tBid and Bcl2 Antagonist X (Bax) proteins and decreased the levels of the anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein. These results suggest that MERL treatment induces mitochondrial dysfunction via decreased levels of Bcl-2 and increased levels of tBid and Bax.

Bottom Line: Also, treatment with MERL increased the activities of caspase-3 and -9 compared with the control.However, inhibitions of p38, extracellular signal regulated kinases (ERKs) and C-Jun N-terminal kinases (JNK) by MERL treatment did not affect cell death.These results suggest that MERL mediated cell death is associated with an intrinsic apoptotic pathway in AGS cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Longevity and Biofunctional Medicine, Healthy Aging Korean Medical Research Center, School of Korean Medicine, Pusan National University, Busan, Korea.

ABSTRACT

Objectives: Rheum undulatum L. has traditionally been used for the treatment of many diseases in Asia. However, its anti-proliferative activity in cancer has still not been studied. In the present study, we investigated the anti-cancer effects of methanol extract of Rheum undulatum L. (MERL) on human adenocarcinoma gastric cell lines (AGS).

Methods: To investigate the anti-cancer effect of MERL on AGS cells, we treated the AGS cells with varying con¬centrations of MERL and performed 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. Cell cycle analyses, measurements of the mitochondrial membrane potential (MMP), caspase activity assays and Western blots were conducted to determine whether AGS cell death occurred by apoptosis.

Results: Treatment with MERL significantly inhibited growth of AGS cells in a concentration dependent manner. MERL treatment in AGS cells leaded to increased accumulation of apoptotic sub G1 phase cells in a concentration dependent manner. In control cultures, 5.38% of the cells were in the sub G1 phase. In MERL treated cells, however, this percentage was significantly increased (9.95% at 70 μg/mL, 15.94% at 140 μg/mL, 26.56% at 210 μg/mL and 38.08% at 280 μg/mL). MERL treatment induced the decreased expression of pro-caspase-8 and -9 in a concentration dependent manner, whereas the expression of the active form of caspase-3 was increased. A subsequent Western blot analysis revealed increased cleaved levels of poly (ADP-ribose) polymerase (PARP) protein. Also, treatment with MERL increased the activities of caspase-3 and -9 compared with the control. MERL treatment increased the levels of the pro-apoptotic truncated Bid (tBid) and Bcl2 Antagonist X (Bax) proteins and decreased the levels of the anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein, whose is the stabilization of mitochondria. However, inhibitions of p38, extracellular signal regulated kinases (ERKs) and C-Jun N-terminal kinases (JNK) by MERL treatment did not affect cell death.

Conclusion: These results suggest that MERL mediated cell death is associated with an intrinsic apoptotic pathway in AGS cells.

No MeSH data available.


Related in: MedlinePlus